SU1315473A1 - Mus musculus mouse hybrid cultivated cells strain used for producing monoclonal antibodies to angiotension-converting ferment of human lungs - Google Patents

Abstract

The invention relates to biotechnology and can be used in biology and experimental medicine. The aim of the invention is to obtain a strain of hybrid cultured mouse mussel Mus musculus, producing monoclonal antibodies to a specific epitope, the human lung angiotensin-converting enzyme (ACE). Strain A-ACE-9B9 is obtained by hybridizing the immune cells of the spleen of BALE mice with myelons X63-A 8-653. The strain is stored in the Specialized collection of transplanted somatic cells of vertebrates of the Institute of Cytology of the Academy of Sciences of the USSR under the number VSKK (P) 28D. The cells are cultured on PPM1-1640 medium with 10% bovine fetal serum and 10% horse serum at 37 C in an atmosphere of 5% COj. The cells are passaged once every 3-4 days, the multiplicity of sieving is 1:10. The secretion of monoclonal antibodies for 3-4 days of cultivation is 15-20 µg / ml of culture fluid and 4-6 mg / ml of ascitic fluid. Monoclonal antibodies belong to the class of IgG (they are used for detecting ACE in tissue sections, cultured cells, biological fluids, etc. 2 tab. § (L with sd CO

Description

I13

The invention relates to biotechnology and can be used in medicine and experimental biology.

The aim of the invention is to obtain a strain of hybrid cultured Mus musculus cells used to obtain monoclonal antibodies to a specific epitope of the human anti-lung enzyme (ACE) enzyme.

Strain was prepared as follows.

The BALB / C line is immunized by intraperitoneal injection of 80 μg of angiotensin-converting enzyme derived from human lung tissue in 0.2 ml of phosphate buffered saline (PBS) containing 30% complete Freund's adjuvant. After 10 days, immunization is repeated by intraperitoneal injection of 60 µg of antigen into EGF without an adjuvant. One week later, the mice were injected with 60 µg of the enzyme intravenously in a volume of 50 µg. Three days after this, 15x10 immune cells of the spleen are hybridized with 5x 10 myeloma cells of X63-Ag8-653 in the presence of a 0.7 ml solution containing 45% (v / v) polyethylene glycol (PEG) mol. mass 3350 and 15% dimethyl sulfoxide for 1 min. After hybridization and washing of cells from PEG, cells are seeded into 96-well panels of 2x10 cells per well. For the cultivation and breeding of hybrids, RPM1-1640 medium is used with the addition of 10% horse and 1% fertile fetal serum, 10 M hypoxanthine, 4x10 M aminonterin, and 1.6x10 M thymidine. Producer hybrids are cloned 2 times by the final dilution method, seeding by the I cell per well, containing 4x10 spleen cells. After the 2nd cloning, almost 100% of the subclones produce antibodies to the angiotensin-converting enzyme. Productive clones denote A-ACE-9B9.

Strain A-ACE-9B9 is stored in the Specialized Collection of Transplanted Samotic Vertebrate Cells of the Institute of Cytology of the Academy of Sciences of the USSR under the number VSKK (P) No. 28 D and has the following features.

Cultural features. Cultivation medium - RPM1-164D medium supplemented with 10% bovine fetal serum and 10% equine

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serum, 4 | UM L-glutamine, 10 yi M pyruvate sodium, 100 µg / ml penicillin, 100 µg / ml streptomycin, enriched with amino acids for basal

Wednesday Needle, vitamins for the environment Needle and O, 05 p M mercaptoethanol.

Cells are cultured at 37 ° C in an atmosphere of 5% CO. A seeding dose of 100 thousand cells / ml, the cells are passaged I times

3-4 days, the multiplicity of sieving 1:10. Suspension culture. The strain produces antibodies for 8 passages in culture.

To grow hybridoma in ascites, cells are introduced into the abdominal cavity of BALB / c mice treated with pristane in an amount of 2-510 cells per mouse. Ascites occurs after 12–16 days. No strain on animals

roar.

The productivity of the strain. The secretion of monoclonal antibodies on the 3-4th day of cultivation is 15-20 µg /

/ ml of culture fluid and 4-6 mg / / ml of ascites fluid.

Characterization of monoclonal antibodies.

Monoclonal antibodies are related

to the class IgGy,. They specifically interact with a specific epitope of the human lung angiotensin-converting enzyme. Specificity is assessed using the ELISA and enrichment test.

Contamination Bacteria and fungi in culture were not detected during long-term observation and culture on nutrient media. Infection with mycoplasma was not detected by staining with dyes on DNA and thymidine label of the culture medium.

Cryoconservation 1-5-10 cells of the strain are preserved in 1 ml of bovine fetal serum with the addition of 10% dimethyl sulfoxide. The temperature is reduced at 4 ° C per minute

to 4 and 1 C per minute to -40 C.

Cells are stored in liquid nitrogen. The viability after thawing is 70-80%.

Example 1. Hybridoma cells are placed in a 25 cm plastic bottle of 5x10 in 5 ml of RPM1-1640 medium with 10% fetal calf serum, 10% horse serum, 4 JU M glutamine, 10 K M sodium pyruvate sodium , 100 µg / ml

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penicillin, 100 μg / ml of streptomycin and 0.05 c M mercaptoethanol. Cells are cultured for 3-4 days when in an atmosphere containing 5% CO. The culture medium is harvested, centrifuged for 800 g and 4 C for J O min. The resulting supernatant is used as a reagent to study the specificity of the interaction of an antibody with the human lung angiotensin-converting enzyme using an ELISA method.

On a polystyrene 96-well microtitre panel, ACE is collected - 1 µg per well in 50 µl of carbonate buffer (100 | C, pH 9.6) at 4 ° C overnight. After the panel was saturated with a 0.2% solution of bovine serum albumin (BSA), 50 µl of the culture medium of strain A-ACE-9B9 was added to the panel cells to reduce the nonspecific binding of antibodies with plastic (for 1 hour at 37 ° C). After that, the panel was washed 4 times with 100 µl of PBS with O, 2% BSA and 0.05% Tween-20, and a conjugate of rabbit antibodies against mouse immunoglobulins was added. Covalently bound to peroxidase (for 1 h at 37 C After the end of the incubation, the immiscible reaction products are washed in this way, and the bound peridase, and therefore the antibodies against ACE, is quantitatively determined by the cleavage of the substrate () in the presence of a chromogen — orthophenylene diamine. The positive reaction is shown by brown staining at 490 nm.

The results of an experiment to study the specificity of the binding of immunoglobulins from the cultured medium of strain A-ACE-9B9 to human ACE of the lungs collected on plastics are presented in Table. 1 (the average of three independent definitions is given).

Table

A-ACE-9B9 1

1:10 1: 100

0.70 0.39

0.25

Continued table.

1: 1000

1:10

one

0.13

0.09 0.07 O „08

5 0 5

0

0

 If, instead of the APF, BSA is adsorbed in the cell, then the amount of immunoglobulins from strain A-ASYO-9B9, cop6: -ipo- nated to BSA and detected by ELISA is at the level of negative control.

The results shown in Table 1 indicate a high specificity of the monoclonal antibody produced by cells of strain A-ACE 9B9 to human ACE of the lungs.

Example 2. Rabbit antibodies against mouse immunoglobulins are adsorbed onto a polystyrene 96-cell panel for microtitre: 100 µl per well at a concentration of 20 µg (poi 4 ° C overnight), then after the plate is loaded with a 0.2% BSA solution in PBS to reduce nonspecific antibody binding by plastic adsorb immunoglobulins from the culture medium from cells of strain A-ACE-EVE, prepared as indicated in the example. After washing the panel O, a 2% solution of BSA in PBS from unbound immunoglobulins is added to the wells in a cell solution (100 µl, enzyme concentration 7 µl / ml). The ACE associated with the monoclonal antibody, produced by strain A-ACE-9B9, is quantified in cells for micro titration using the fluorescent method, using Hip-His-Leu as a substrate.

The results of the experiment on the study of the specificity of the interaction of MCAT, produced by cells of strain A-ACE-9B9 with ACF in solution, are presented in Table. 2 (the average of three independent definitions is given).

five

T a b

persons 2

one

eleven

eleven;

one

ten

100

1000

ten

7.82 6.37 2.77 0.84 0.330

0.340

0.324

Compiled by M. Serov

Editor N.Egorova1 HE B B 9R1 .. Proofreader L. Pa Tai

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VNIIPI USSR State Committee

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1315473

The results in Table 2 indicate the high specificity of monoconal antibodies produced by cells of strain A-ACE-9B9 with respect to ACE, and show the possibility of their use for detecting ACE in solutions (in biological fluids, tissue extracts, etc.) d.)

GO f

o rm u la invention

The strain of hybrid cultured mouse cells Mus musculus VASK (P) No. 28D (a specialized collection of transplanted somatic cells of the vertebrates of the Institute of Cytology of the Academy of Sciences of the USSR) used to produce monoclonal antibodies to angiotensin-converting human lung enzyme.

Claims (1)

The strain of hybrid cultured mouse cells Mus muscuius VSCC (Π) No. 28D (specialized collection of 15 transplantable somatic vertebrate cells of the Institute of Cytology of the USSR Academy of Sciences) ,! Used to obtain monoclonal antibodies to angiotensin-converting human lung enzyme.